Antibodies against cancer antigen TMEFF2 and uses thereof

ABSTRACT

Described herein are methods and compositions that can be used for diagnosis and treatment of cancer.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Ser. No. 60/362,837,filed Mar. 8, 2002, and U.S. Ser. No. 60/436,812, filed Dec. 27, 2002,each of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to the identification and generation ofantibodies that specifically bind to TMEFF2 proteins that are involvedin cancer; and to the use of such antibodies and compositions comprisingthem in the diagnosis, prognosis and therapy of cancer.

BACKGROUND OF THE INVENTION

[0003] Prostate cancer is the most frequently diagnosed cancer and thesecond leading cause of male cancer death in North America and northernEurope. Early detection of prostate cancer using a serum test forprostate-specific antigen (PSA) has dramatically improved the treatmentof the disease (Oesterling, 1992, J. Am. Med. Assoc. 267:2236-2238 andDiVita et al. (1997) Cancer: Principles and Practices of Oncology, 5thed. Lippincott-Raven pub.). Treatment of prostate cancer consistslargely of surgical prostatectomy, radiation therapy, androgen ablationtherapy and chemotherapy. Although many prostate cancer patients areeffectively treated, the current therapies can all induce serious sideeffects which diminish quality of life. For example, patients whopresent with metastatic disease are most often treated withandrogen-ablation therapy. Chemical or surgical castration has been theprimary treatment for symptomatic metastatic prostate cancer for over 50years. While this testicular androgen deprivation therapy usuallyresults in stabilization or regression of the disease (in 80% ofpatients), progression of metastatic prostate cancer eventually develops(Panvichian et al., Cancer Control 3(6):493-500 (1996); Afrin andStuart, 1994, J. S. C. Med. Assoc. 90:231-236). Metastatic disease iscurrently considered incurable. Thus, the primary goals of treatment areto prolong survival and improve quality of life (Rago, Cancer Control5(6):513-521 (1998)).

[0004] Clearly, the identification of novel therapeutic targets anddiagnostic markers is essential for improving the current treatment ofprostate cancer patients. Recent advances in molecular medicine haveincreased the interest in tumor-specific cell surface antigens thatcould serve as targets for various immunotherapeutic or small moleculestrategies. Antigens suitable for immunotherapeutic strategies should behighly expressed in cancer tissues and ideally not expressed in normaladult tissues. One such antigen is TMEFF2.

[0005] The TMEFF2 protein contains 2 follistatin-like domains and aconserved EGF-like domain. The gene encoding the protein was firstcharacterized from a human brain cDNA library (see Uchida, et al. (1999)Biochem. Biophys. Res. Commun. 266:593-602), and later isolated from ahuman fetal brain cDNA library (see Horie, et al. (2000) Genomics67:146-152). See also, e.g., Online Mendelian Inheritance in Man, number605734; Unigene Cluster Hs.22791; LocusLink 23671; and other linkedsites. TMEFF2 has been referred to as tomoregulin, TR, hyperplasticpolyposis gene 1, HPP 1, and TENB2. TMEFF2's nucleic acid sequence canbe identified by ATCC Accession Nos. AF264150, AB004064, AB017269, andAF179274. TMEFF2's amino acid sequence can be identified by ATCCAccession Nos. AAF91397, BAA90820, BAA87897, and AAD55776. TMEFF2'sUniGene Cluster identification number is hs.22791, Locuslinkidentification number is 23671, and OMIM identification number is605734.

[0006] The gene has also been implicated in certain cancerousconditions. Young, et al. (2001) Proc. Nat'l Acad. Sci. USA 98:265-270reported expression in colorectal polyps. Glynne-Jones, et al. (2001)Int. J. Cancer 94:178-184 reported it as a marker for prostate cancer.

[0007] Treatments such as surgery, radiation therapy, and cryotherapyare potentially curative when the cancer remains localized. Therefore,early detection of cancer is important for a positive prognosis fortreatment.

[0008] Thus, antibodies that can be used for diagnosis and prognosis andeffective treatment of cancer, and including particularly metastaticcancer, would be desirable. Accordingly, provided herein arecompositions and methods that can be used in diagnosis, prognosis, andtherapy of certain cancers.

SUMMARY OF THE INVENTION

[0009] The present invention provides anti-TMEFF2 antibodies that aresurprisingly well internalized and are particularly useful for makingconjugated antibodies for therapeutic purposes. In some embodiments, theantibodies of the present invention are therapeutically useful inpersons diagnosed with cancer and other proliferative conditions,including benign proliferative conditions. In one aspect, the antibodiesof the present invention can be used to treat proliferative conditionsof the prostate including, e.g., benign prostate hyperplasia andprostate cancer. In another aspect, the antibodies of the presentinvention can be used to treat malignant and benign proliferativeconditions of the brain including, e.g., gliobastomas,oligodendrogliomas, anablastic astrocytomas, meningiomas,medulloblastomas, and neuroblastomas.

[0010] In particular, the present invention provides anti-TMEFF2antibodies that are particularly useful as selective cytotoxic agentsfor TMEFF2 expressing cells. Without wishing to be bound by theory it isbelieved that the antibodies of the invention recognize a TMEFF2 epitopethat effects an increased internalization, and thus enhanced cellkilling, when conjugated to a cytotoxic moiety.

[0011] The present invention provides antibodies that competitivelyinhibit binding of TMEFF2#19 (ATCC Accession No. PTA-4127) to TMEFF2. Insome embodiments the antibodies are further conjugated to an effectorcomponent. The effector component can be a label (e.g., a fluorescentlabel) or can be cytotoxic moiety (e.g., a radioisotope or a cytotoxicchemical) An exemplary cytotoxic chemical is auristatin.

[0012] The antibodies of the invention can be whole antibodies or canantibody fragments. In some embodiments the immunoglobulin is ahumanized antibody. An exemplary antibody of the invention is TMEFF2#19(ATCC Accession No. PTA-4127).

[0013] The invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable excipient and the antibody ofthe invention. In these embodiments, the antibody can be furtherconjugated to an effector component. The effector component can be alabel (e.g., a fluorescent label) or can be cytotoxic moiety (e.g., aradioisotope or a cytotoxic chemical) An exemplary cytotoxic chemical isauristatin. The antibodies in the pharmaceutical compositions can bewhole antibodies or can antibody fragments. In some embodiments theimmunoglobulin is a humanized antibody. An exemplary antibody TMEFF2#19(ATCC Accession No. PTA-4127).

[0014] The invention further provides immunoassays using theimmunoglobulins of the invention. These methods involve detecting aprostate cancer cell in a biological sample from a patient by contactingthe biological sample with an antibody of the invention. The antibody istypically conjugated to a label such as fluorescent label.

[0015] The invention provides methods of inhibiting proliferation of aprostate cancer-associated cell. The method comprises contacting thecell with an antibody of the invention. In most embodiments, the cancercell is in a patient, typically a human. The patient may be undergoing atherapeutic regimen to treat metastatic prostate cancer or may besuspected of having prostate cancer. The invention also provides amethod of treating prostate cancer with an antibody to TMEFF2, whereinsaid prostate cancer is selected from the group consisting of a primaryprostate cancer, metastatic prostate cancer, locally advanced prostatecancer, androgen independent prostate cancer, prostate cancer that hasbeen treated with neoadjuvant therapy, and prostate cancer that isrefractory to treatment with neoadjuvant therapy.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides novel reagents and methods fortreatment, diagnosis and prognosis for certain cancers using antibodiesagainst TMEFF2. In particular, the present invention providesanti-TMEFF2 antibodies that are particularly useful as selectivecytotoxic agents for TMEFF2 expressing cells. Without wishing to bebound by theory it is believed that the antibodies of the inventionrecognize a TMEFF2 epitope that effects an increased internalization andthus enhanced cell killing, when conjugated to a cytotoxic moiety. Inaddition, antibodies of the invention are useful because they recognizethe non-glycosylated form of the protein. This is advantageous becauseantibodies that recognize the glycosylated portion of the protein mayonly recognize a subset of the expressed proteins. The invention isbased, in part, on analysis of approximately 100 hybridoma supernatants.Epitope mapping of antibodies showing high affinity binding was carriedout through competitive binding analyses. Using this methodologyantibodies recognizing a number of individual epitopes were identified.The antibodies were then assessed for TMEFF2 dependent cell death invitro. Using these methods antibodies that promoted significant celldeath were identified.

Definitions

[0017] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene, or fragments thereof, that specificallybinds and recognizes an antigen. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon, and mu constantregion genes, as well as the myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. Typically, the antigen-binding region of an antibody orits functional equivalent will be most critical in specificity andaffinity of binding. See Paul, Fundamental Immunology. However,recombinant methods exist to chimerize and generate changed classes andeffector functions.

[0018] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer of four polypeptides. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus ofeach chain defines a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. The terms variablelight chain (VL) and variable heavy chain (VH) refer to these light andheavy chains respectively.

[0019] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, e.g., pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab′)₂, a dimer of Fab whichitself is a light chain joined to VH-CH1 by a disulfide bond. TheF(ab′)₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab′)₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty, et al. (1990) Nature348:552-554).

[0020] For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein (1975) Nature 256:495-497; Kozbor, et al. (1983)Immunology Today 4:72; Cole, et al., pp. 77-96 in Monoclonal Antibodiesand Cancer Therapy (1985); Coligan (1991) Current Protocols inImmunology; Harlow & Lane (1988) Antibodies: A Laboratory Manual; andGoding(1986) Monoclonal Antibodies: Principles and Practice (2d ed.).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty, et al. (1990) Nature 348:552-554; Marks, et al.(1992) Biotechnology 10:779-783).

[0021] A “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

[0022] “Epitope” or “antigenic determinant” refers to a site on anantigen to which an antibody binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., “Epitope Mapping Protocols” in Morris(ed. 1996) Methods in Molecular Biology, Vol. 66.

[0023] The term “TMEFF2 protein” or “TMEFF2 polynucleotide” refers tonucleic acid and polypeptide polymorphic variants, alleles, mutants, andinterspecies homologues that: (1) have a nucleotide sequence that hasgreater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%,85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% orgreater nucleotide sequence identity, preferably over a region of over aregion of at least about 25, 50, 100, 200, 500, 1000, or morenucleotides, to a nucleotide sequence of SEQ ID NO:1; (2) bind toantibodies, e.g., polyclonal antibodies, raised against an immunogencomprising an amino acid sequence encoded by a nucleotide sequence ofSEQ ID NO: 1, and conservatively modified variants thereof, (3)specifically hybridize under stringent hybridization conditions to anucleic acid sequence, or the complement thereof of SEQ ID NO: 1 andconservatively modified variants thereof or (4) have an amino acidsequence that has greater than about 60% amino acid sequence identity,65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% or greater amino sequence identity, preferably over aregion of at least about 25, 50, 100, 200, or more amino acids, to anamino acid sequence of SEQ ID NO:2. A polynucleotide or polypeptidesequence is typically from a mammal including, but not limited to,primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig,horse, sheep, or other mammal. A “TMEFF2 polypeptide” and a “TMEFF2polynucleotide,” include both naturally occurring or recombinant forms.A number of different variants have been identified. See, e.g.,LocusLink 23671.

[0024] A “full length” TMEFF2 protein or nucleic acid refers to aprostate cancer polypeptide or polynucleotide sequence, or a variantthereof, that contains all of the elements normally contained in one ormore naturally occurring, wild type TMEFF2 polynucleotide or polypeptidesequences. For example, a full length TMEFF2 nucleic acid will typicallycomprise all of the exons that encode for the full length, naturallyoccurring protein. The “full length” may be prior to, or after, variousstages of post-translation processing or splicing, including alternativesplicing.

[0025] “Biological sample” as used herein is a sample of biologicaltissue or fluid that contains nucleic acids or polypeptides, e.g., of aTMEFF2 protein, polynucleotide or transcript. Such samples include, butare not limited to, tissue isolated from primates, e.g., humans, orrodents, e.g., mice, and rats. Biological samples may also includesections of tissues such as biopsy and autopsy samples, frozen sectionstaken for histologic purposes, blood, plasma, serum, sputum, stool,tears, mucus, hair, skin, etc. Biological samples also include explantsand primary and/or transformed cell cultures derived from patienttissues. A biological sample is typically obtained from a eukaryoticorganism, most preferably a mammal such as a primate, e.g., chimpanzeeor human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit;or a bird; reptile; or fish.

[0026] “Providing a biological sample” means to obtain a biologicalsample for use in methods described in this invention. Most often, thiswill be done by removing a sample of cells from an animal, but can alsobe accomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods of the invention in vivo. Archival tissues,having treatment or outcome history, will be particularly useful.

[0027] The term “prostate cancer stage” or grammatical equivalentsthereof refer to the size of a cancer and whether it has spread beyondits original site. Prostate cancer is generally divided into fourstages, from small and localized (stage 1), to spread into surroundingtissue (stage 3 and 4). If the cancer has spread to other parts of thebody, this is known as secondary prostate cancer (or metastatic prostatecancer). There are two systems of prostate cancer staging theconventional system of the American Urological Association and a newsystem based on detection-of prostate cancer by way of prostate serumantigen (PSA) tests. The new system known as the Tumor, Nodes andMetastasis System or TNM. In the conventional AUA system stage Acorresponds to clinically unsuspected prostate cancer. Stage Bcorresponds to a tumor confined to the prostate gland (localized). StageC corresponds to a tumor outside prostate capsule, and stage Dcorresponds to metastasis into the pelvic lymph node. Stage D2 isdistant metastatic cancer into distant lymph nodes, organs, soft tissueor bone.

[0028] In the TNM system stages include T1: The tumor is within theprostate gland and is too small to be detected during a rectalexamination, but may be detected through tests such as PSA test. Thereare generally no symptoms. T2: The tumor is still within the prostategland but is large enough to be felt during a digital rectal examinationor show up on ultrasound. Often there are no symptoms. T3/T4: The cancerhas spread beyond the prostate gland into the surrounding tissues. Thisis known as locally advanced prostate cancer. T1 and T2 tumors are knownas early prostate cancer. T3 and T4 are known as locally advancedprostate cancer. If the lymph nodes, bones or other parts of the bodyare affected this is called secondary or metastatic cancer. “Locallyadvanced prostate cancer” refers to prostate cancer that shows someevidence of metastasis, or developing metastasis.

[0029] The term “neoadjuvant therapy” also known as “neoadjuvantandrogen depravation therapy” refers to the treatment of prostate cancerby giving adjuvant hormone blocking drugs before surgery.

[0030] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions, aswell as naturally occurring, e.g., polymorphic or allelic variants, andman-made variants. As described below, the preferred algorithms canaccount for gaps and the like. Preferably, identity exists over a regionthat is at least about 25 amino acids or nucleotides in length, or morepreferably over a region that is 50-100 amino acids or nucleotides inlength.

[0031] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0032] A “comparison window”, as used herein, includes reference to asegment of one of the number of contiguous positions selected from thegroup consisting typically of from about 20 to 600, usually about 50 toabout 200, more usually about 100 to about 150 in which a sequence maybe compared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. Methods ofalignment of sequences for comparison are well-known in the art. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith & Waterman (1981) Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman & Wunsch (1970)J. Mol. Biol. 48:443, by the search for similarity method of Pearson &Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Ausubel, et al. (eds. 1995 and supplements)Current Protocols in Molecular Biology.

[0033] Preferred examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity includethe BLAST and BLAST 2.0 algorithms, which are described in Altschul, etal. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul, et al. (1990) J.Mol. Biol. 215:403-410. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, e.g.,for nucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0034] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul (1993)Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. Log valuesmay be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110,150, 170, etc.

[0035] An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, e.g., where the two peptides differonly by conservative substitutions. Another indication that two nucleicacid sequences are substantially identical is that the two molecules ortheir complements hybridize to each other under stringent conditions, asdescribed below. Yet another indication that two nucleic acid sequencesare substantially identical is that the same primers can be used toamplify the sequences.

[0036] A “host cell” is a naturally occurring cell or a transformed cellthat contains an expression vector and supports the replication orexpression of the expression vector. Host cells may be cultured cells,explants, cells in vivo, and the like. Host cells may be prokaryoticcells such as E. coli, or eukaryotic cells such as yeast, insect,amphibian, or mammalian cells such as CHO, HeLa, and the like (see,e.g., the American Type Culture Collection catalog or web site,www.atcc.org).

[0037] The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein or nucleic acid that is thepredominant species present in a preparation is substantially purified.In particular, an isolated nucleic acid is separated from some openreading frames that naturally flank the gene and encode proteins otherthan protein encoded by the gene. The term “purified” in someembodiments denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure. “Purify” or“purification” in other embodiments means removing at least onecontaminant from the composition to be purified. In this sense,purification does not require that the purified compound be homogenous,e.g., 100% pure.

[0038] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

[0039] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

[0040] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0041] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, often silent variations of a nucleicacid which encodes a polypeptide is implicit in a described sequencewith respect to the expression product, but not with respect to actualprobe sequences.

[0042] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention. Typicallyconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Crceighton (1984) Proteins).

[0043] Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts, et al. (1994)Molecular Biology of the Cell (3d ed.), and Cantor & Schimmel (1980)Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules. “Primary structure” refers to the amino acid sequence ofa particular peptide. “Secondary structure” refers to locally ordered,three dimensional structures within a polypeptide. These structures arecommonly known as domains. Domains are portions of a polypeptide thatoften form a compact unit of the polypeptide and are typically 25 toapproximately 500 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed, usually by the noncovalentassociation of independent tertiary units. Anisotropic terms are alsoknown as energy terms.

[0044] A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include fluorescentdyes, electron-dense reagents, enzymes (e.g., as commonly used in anELISA), biotin, digoxigenin, or haptens and proteins or other entitieswhich can be made detectable, e.g., by incorporating a radiolabel intothe peptide or used to detect antibodies specifically reactive with thepeptide. The radioisotope may be, for example, ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I. In some cases, particularly using antibodies against the proteinsof the invention, the radioisotopes are used as toxic moieties, asdescribed below. The labels may be incorporated into the TMEFF2 nucleicacids, proteins and antibodies at any position. A method known in theart for conjugating the antibody to the label may be employed, includingthose methods described by Hunter, et al. (1962) Nature 144:945; David,et al. (1974) Biochemistry 13:1014; Pain, et al. (1981) J. Immunol.Meth. 40:219; and Nygren (1982) J. Histochem. and Cytochem. 30:407. Thelifetime of radiolabeled peptides or radiolabeled antibody compositionsmay extended by the addition of substances that stabilize theradiolabeled peptide or antibody and protect it from degradation. Anysubstance or combination of substances that stabilize the radiolabeledpeptide or antibody may be used including those substances disclosed inU.S. Pat. No. 5,961,955.

[0045] An “effector” or “effector moiety” or “effector component” is amolecule that is bound (or linked, or conjugated), either covalently,through a linker or a chemical bond, or noncovalently, through ionic,van der Waals, electrostatic, or hydrogen bonds, to an antibody. The“effector” can be a variety of molecules including, e.g., detectionmoieties including radioactive compounds, fluorescent compounds, anenzyme or substrate, tags such as epitope tags, a toxin; activatablemoieties, a chemotherapeutic agent; a lipase; an antibiotic; or aradioisotope emitting “hard”, e.g., beta radiation.

[0046] The term “recombinant” when used with reference, e.g., to a cell,or nucleic acid, protein, or vector, indicates that the cell, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, e.g., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, e.g., throughthe expression of a recombinant nucleic acid as depicted above.

[0047] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

[0048] A “promoter” is defined as an array of nucleic acid controlsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

[0049] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0050] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein, in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein sequences at least two times the background and more typicallymore than 10 to 100 times background.

[0051] Specific binding to an antibody under such conditions requires anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to a particular protein,polymorphic variants, alleles, orthologs, and conservatively modifiedvariants, or splice variants, or portions thereof, can be selected toobtain only those polyclonal antibodies that are specificallyimmunoreactive with TMEFF2 and not with other proteins. This selectionmay be achieved by subtracting out antibodies that cross-react withother molecules. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane (1988) Antibodies: A Laboratory Manual for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

[0052] “Tumor cell” refers to precancerous, cancerous, and normal cellsin a tumor.

[0053] “Cancer cells,” “transformed” cells or “transformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation is associated withphenotypic changes, such as immortalization of cells, aberrant growthcontrol, nonmorphological changes, and/or malignancy (see, Freshney(1994) Culture of Animal Cells: A Manual of Basic Technique (3d ed.).

Expression of TMEFF2 Polypeptides from Nucleic Acids

[0054] Nucleic acids of the invention can be used to make a variety ofexpression vectors to express TMEFF2 polypeptides which can then be usedto raise antibodies of the invention, as described below. Expressionvectors and recombinant DNA technology are well known to those of skillin the art (see, e.g., Ausubel, supra, and Fernandez & Hoeffler (eds.1999) Gene Expression Systems) and are used to express proteins. Theexpression vectors may be either self-replicating extrachromosomalvectors or vectors which integrate into a host genome. Generally, theseexpression vectors include transcriptional and translational regulatorynucleic acid operably linked to the nucleic acid encoding the TMEFF2protein. The term “control sequences” refers to DNA sequences used forthe expression of an operably linked coding sequence in a particularhost organism. Control sequences that are suitable for prokaryotes,e.g., include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0055] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis typically accomplished by ligation at convenient restriction sites.If such sites do not exist, synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice.Transcriptional and translational regulatory nucleic acid will generallybe appropriate to the host cell used to express the TMEFF2 protein.Numerous types of appropriate expression vectors, and suitableregulatory sequences are known in the art for a variety of host cells.

[0056] In general, transcriptional and translational regulatorysequences may include, but are not limited to, promoter sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, and enhancer or activatorsequences. In a preferred embodiment, the regulatory sequences include apromoter and transcriptional start and stop sequences.

[0057] Promoter sequences encode either constitutive or induciblepromoters. The promoters may be either naturally occurring promoters orhybrid promoters. Hybrid promoters, which combine elements of more thanone promoter, are also known in the art, and are useful in the presentinvention.

[0058] In addition, an expression vector may comprise additionalelements. For example, the expression vector may have two replicationsystems, thus allowing it to be maintained in two organisms, e.g., inmammalian or insect cells for expression and in a prokaryotic host forcloning and amplification. Furthermore, for integrating expressionvectors, the expression vector contains at least one sequence homologousto the host cell genome, and preferably two homologous sequences whichflank the expression construct. The integrating vector may be directedto a specific locus in the host cell by selecting the appropriatehomologous sequence for inclusion in the vector. Constructs forintegrating vectors are well known in the art (e.g., Fernandez &Hoeffler, supra).

[0059] In addition, in a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selection genes are well known in the art and will vary withthe host cell used.

[0060] The TMEFF2 proteins of the present invention are produced byculturing a host cell transformed with an expression vector containingnucleic acid encoding a TMEFF2 protein, under the appropriate conditionsto-induce or cause expression of the TMEFF2 protein. Conditionsappropriate for TMEFF2 protein expression will vary with the choice ofthe expression vector and the host cell, and will be easily ascertainedby one skilled in the art through routine experimentation oroptimization. For example, the use of constitutive promoters in theexpression vector will require optimizing the growth and proliferationof the host cell, while the use of an inducible promoter requires theappropriate growth conditions for induction. In addition, in someembodiments, the timing of the harvest is important. For example, thebaculoviral systems used in insect cell expression are lytic viruses,and thus harvest time selection can be crucial for product yield.

[0061] Appropriate host cells include yeast, bacteria, archaebacteria,fungi, and insect and animal cells, including mammalian cells. Ofparticular interest are Saccharomyces cerevisiae and other yeasts, E.coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora,BHK, CHO, COS, HeLa cells, HUVEC (human umbilical vein endothelialcells), THP1 cells (a macrophage cell line) and various other humancells and cell lines.

[0062] In a preferred embodiment, the TMEFF2 proteins are expressed inmammalian cells. Mammalian expression systems are also known in the art,and include retroviral and adenoviral systems. One expression vectorsystem is a retroviral vector system such as is generally described inPCT/US97/01019 and PCT/US97/01048, both of which are hereby expresslyincorporated by reference. Of particular use as mammalian promoters arethe promoters from mammalian viral genes, since the viral genes areoften highly expressed and have a broad host range. Examples include theSV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirusmajor late promoter, herpes simplex virus promoter, and the CMV promoter(see, e.g., Fernandez & Hoeffler, supra). Typically, transcriptiontermination and polyadenylation sequences recognized by mammalian cellsare regulatory regions located 3′ to the translation stop codon andthus, together with the promoter elements, flank the coding sequence.Examples of transcription terminator and polyadenylation signals includethose derived form SV40.

[0063] The methods of introducing exogenous nucleic acid into mammalianhosts, as well as other hosts, is well known in the art, and will varywith the host cell used. Techniques include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, viral infection,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei.

[0064] In some embodiments, TMEFF2 proteins are expressed in bacterialsystems. Bacterial expression systems are well known in the art.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;e.g., the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription. In addition toa functioning promoter sequence, an efficient ribosome binding site isdesirable. The expression vector may also include a signal peptidesequence that provides for secretion of the TMEFF2 protein in bacteria.The protein is either secreted into the growth media (gram-positivebacteria) or into the periplasmic space, located between the inner andouter membrane of the cell (gram-negative bacteria). The bacterialexpression vector may also include a selectable marker gene to allow forthe selection of bacterial strains that have been transformed. Suitableselection genes include genes which render the bacteria resistant todrugs such as ampicillin, chloramphenicol, erythromycin, kanamycin,neomycin and tetracycline. Selectable markers also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways. These components are assembled into expressionvectors. Expression vectors for bacteria are well known in the art, andinclude vectors for Bacillus subtilis, E. coli, Streptococcus cremoris,and Streptococcus lividans, among others (e.g., Fernandez & Hoeffler,supra). The bacterial expression vectors are transformed into bacterialhost cells using techniques well known in the art, such as calciumchloride treatment, electroporation, and others.

[0065] In one embodiment, TMEFF2 polypeptides are produced in insectcells. Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

[0066] TMEFF2 polypeptides can also be produced in yeast cells. Yeastexpression systems are well known in the art, and include expressionvectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa,Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichiaguillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowialipolytica.

[0067] The TMEFF2 polypeptides may also be made as a fusion protein,using techniques well known in the art. Thus, e.g., for the creation ofmonoclonal antibodies, if the desired epitope is small, the TMEFF2protein may be fused to a carrier protein to form an immunogen.Alternatively, the TMEFF2 protein may be made as a fusion protein toincrease expression, or for other reasons. For example, when the TMEFF2protein is a TMEFF2 peptide, the nucleic acid encoding the peptide maybe linked to other nucleic acid for expression purposes.

[0068] The TMEFF2 polypeptides are typically purified or isolated afterexpression. TMEFF2 proteins may be isolated or purified in a variety ofways known to those skilled in the art depending on what othercomponents are present in the sample. Standard purification methodsinclude electrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the TMEFF2 protein may be purified using a standard anti-TMEFF2 proteinantibody column. Ultrafiltration and diafiltration techniques, inconjunction with protein concentration, are also useful. For generalguidance in suitable purification techniques, see Scopes, ProteinPurification (1982). The degree of purification necessary will varydepending on the use of the TMEFF2 protein. In some instances nopurification will be necessary.

[0069] One of skill will recognize that the expressed protein need nothave the wild-type TMEFF2 sequence but may be derivative or variant ascompared to the wild-type sequence. These variants typically fall intoone or more of three classes: substitutional, insertional or deletionalvariants. These variants ordinarily are prepared by site specificmutagenesis of nucleotides in the DNA encoding the protein, usingcassette or PCR mutagenesis or other techniques well known in the art,to produce DNA encoding the variant, and thereafter expressing the DNAin recombinant cell culture as outlined above. However, variant proteinfragments having up to about 100-150 residues may be prepared by invitro synthesis using established techniques. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the TMEFF2 protein amino acid sequence. Thevariants typically exhibit the same qualitative biological activity asthe naturally occurring analogue, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

[0070] TMEFF2 polypeptides of the present invention may also be modifiedin a way to form chimeric molecules comprising a TMEFF2 polypeptidefused to another, heterologous polypeptide or amino acid sequence. Inone embodiment, such a chimeric molecule comprises a fusion of theTMEFF2 polypeptide with a tag polypeptide which provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino-or carboxyl-terminus of the TMEFF2polypeptide. The presence of such epitope-tagged forms of a TMEFF2polypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the TMEFF2polypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of a TMEFF2 polypeptide with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule.

[0071] Various tag polypeptides and their respective antibodies are wellknown in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; HIS6 and metal chelationtags, the flu HA tag polypeptide and its antibody 12CA5 (Field, et al(1988) Mol. Cell. Biol. 8:2159-2165); the c-myc tag and the 8F9, 3C7,6E10, G4, B7 and 9E10 antibodies thereto (Evan, et at. (1985) Molecularand Cellular Biology 5:3610-3616); and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody (Paborsky, et al. (1990)Protein Engineering 3(6):547-553). Other tag polypeptides include theFLAG-peptide (Hopp, et al. (1988) BioTechnology 6:1204-1210); the KT3epitope peptide (Martin, et al. (1992) Science 255:192-194); tubulinepitope peptide (Skinner, et al. (1991) J. Biol. Chem. 266:15163-15166);and the T7 gene 10 protein peptide tag (Lutz-Freyermnuth, et al. (1990)Proc. Natl. Acad. Sci. USA 87:6393-6397).

Antibodies to Cancer Proteins

[0072] Once the TMEFF2 protein is produced, it is used to generateantibodies, e.g., for immunotherapy or immunodiagnosis. As noted above,the antibodies of the invention recognize the same epitope as thatrecognized by TMEFF2#19 (ATCC Accession No. PTA-4127). The ability of aparticular antibody to recognize the same epitope as another antibody istypically determined by the ability of one antibody to competitivelyinhibit binding of the second antibody to the antigen. Many of a numberof competitive binding assays can be used to measure competition betweentwo antibodies to the same antigen. An exemplary assay is a Biacoreassay as desrcibed in the Examples, below. Briefly in these assays,binding sites can be mapped in structural terms by testing the abilityof interactants, e.g. different antibodies, to inhibit the binding ofanother. Injecting two consecutive antibody samples in sufficientconcentration can identify pairs of competing antibodies for the samebinding epitope. The antibody samples should have the potential to reacha significant saturation with each injection. The net binding of thesecond antibody injection is indicative for binding epitope analysis.Two response levels can be used to describe the boundaries of perfectcompetition versus non-competing binding due to distinct epitopes. Therelative amount of binding response of the second antibody injectionrelative to the binding of identical and distinct binding epitopesdetermines the degree of epitope overlap.

[0073] Other conventional immunoassays known in the art can be used inthe present invention. For example, antibodies can be differentiated bythe epitope to which they bind using a sandwich ELISA assay. This iscarried out by using a capture antibody to coat the surface of a well. Asubsaturating concentration of tagged-antigen is then added to thecapture surface. This protein will be bound to the antibody through aspecific antibody:epitope interaction. After washing a second antibody,which has been covalently linked to a detectable moiety (e.g., HRP, withthe labeled antibody being defined as the detection antibody) is addedto the ELISA. If this antibody recognizes the same epitope as thecapture antibody it will be unable to bind to the target protein as thatparticular epitope will no longer be available for binding. If howeverthis second antibody recognizes a different epitope on the targetprotein it will be able to bind and this binding can be detected byquantifying the level of activity (and hence antibody bound) using arelevant substrate. The background is defined by using a single antibodyas both capture and detection antibody, whereas the maximal signal canbe established by capturing with an antigen specific antibody anddetecting with an antibody to the tag on the antigen. By using thebackground and maximal signals as references, antibodies can be assessedin a pair-wise manner to determine epitope specificity.

[0074] A first-antibody is considered to competitively inhibit bindingof a second antibody, if binding of the second antibody to the antigenis reduced by at least 30%, usually at least about 40%, 50%, 60% or 75%,and often by at least about 90%, in the presence of the first antibodyusing any of the assays described above.

[0075] Methods of preparing polyclonal antibodies are known to theskilled artisan (e.g., Coligan, supra; and Harlow & Lane, supra).Polyclonal antibodies can be raised in a mammal, e.g., by one or moreinjections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include a protein encoded by a nucleic acid of thefigures or fragment thereof or a fusion protein thereof. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include but are not limited to keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examplesof adjuvants which may be employed include Freund's complete adjuvantand MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

[0076] The antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler & Milstein (1975) Nature 256:495. In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro. The immunizing agent will typically include apolypeptide encoded by a nucleic acid of Tables 1-2, fragment thereof,or a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell (pp. 59-103 in Goding (1986) Monoclonal Antibodies:Principles and Practice). Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine, and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

[0077] In one embodiment, the antibodies are bispecific antibodies.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens or that have binding specificities for two epitopes on the sameantigen. In one embodiment, one of the binding specificities is for aTMEFF2 protein, the other one is for any other prostate cancer antigen.Alternatively, tetramer-type technology may create multivalent reagents.

[0078] In a preferred embodiment, the antibodies to TMEFF2 protein arecapable of reducing or eliminating prostate cancer cells. That is, theaddition of anti-TMEFF2 antibodies (either polyclonal or preferablymonoclonal) to prostate cancer tissue (or cells containing TMEFF2) mayreduce or eliminate the prostate cancer. Generally, at least a 25%decrease in activity, growth, size or the like is preferred, with atleast about 50% being particularly preferred and about a 95-100%decrease being especially preferred.

[0079] In a preferred embodiment the antibodies to the TMEFF2 proteinsare humanized antibodies (e.g., Xenerex Biosciences, Medarex, Inc.,Abgenix, Inc., Protein Design Labs, Inc.) Humanized forms of non-human(e.g., murine) antibodies are chimeric molecules of immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, selectivity, affinity, and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, a humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the framework (FR) regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region(Fe), typically that of a human immunoglobulin (Jones, et al. (1986)Nature 321:522-525; Riechmann, et al. (1988) Nature 332:323-329; andPresta (1992) Curr. Op. Struct. Biol. 2:593-596). Humanization can beessentially performed following the method of Winter and co-workers(Jones, et al. (1986) Nature 321:522-525; Riechmann, et al. (1988)Nature 332:323-327; Verhoeyen, et al. (1988) Science 239:1534-1536), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a nonhuman species.

[0080] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries (Hoogenboom & Winter(1991) J. Mol. Biol. 227:381; Marks, et al. (1991) J. Mol. Biol.222:581). The techniques of Cole, et al. and Boemer, et al. are alsoavailable for the preparation of human monoclonal antibodies (p. 77 inCole, et al. (1985) Monoclonal Antibodies and Cancer Therapy; andBoerner, et al. (1991) J. Immunol. 147(l):86-95). Similarly, humanantibodies can be made by introducing of human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, e.g., in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks, et al. (1992)Bio/Technology 10:779-783; Lonberg, et al. (1994) Nature 368:856-859;Morrison (1994) Nature 368:812-13; Fishwild, et al. (1996) NatureBiotechnology 14:845-51; Neuberger (1996) Nature Biotechnology 14:826;and Lonberg & Huszar (1995) Intern. Rev. Immunol. 13:65-93.

[0081] By immunotherapy is meant treatment of prostate cancer with anantibody raised against TMEFF2 proteins. As used herein, immunotherapycan be passive or active. Passive immunotherapy as defined herein is thepassive transfer of antibody to a recipient (patient). Activeimmunization is the induction of antibody and/or T-cell responses in arecipient (patient). Induction of an immune response is the result ofproviding the recipient with an antigen to which antibodies are raised.As appreciated by one of ordinary skill in the art, the antigen may beprovided by injecting a polypeptide against which antibodies are desiredto be raised into a recipient, or contacting the recipient with anucleic acid capable of expressing the antigen and under conditions forexpression of the antigen, leading to an immune response.

[0082] In some embodiments, the antibody is conjugated to an effectormoiety. The effector moiety can be any number of molecules, includinglabeling moieties such as radioactive labels or fluorescent labels, orcan be a therapeutic moiety. In one aspect the therapeutic moiety is asmall molecule that modulates the activity of the TMEFF2 protein. Inanother aspect the therapeutic moiety modulates the activity ofmolecules associated with or in close proximity to the TMEFF2 protein.

[0083] In other embodiments, the therapeutic moiety is a cytotoxicagent. In this method, targeting the cytotoxic agent to prostate cancertissue or cells, results in a reduction in the number of afflictedcells, thereby reducing symptoms associated with prostate cancer.Cytotoxic agents are numerous and varied and include, but are notlimited to, cytotoxic drugs or toxins or active fragments of suchtoxins. Suitable toxins and their corresponding fragments includediphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain,curcin, crotin, phenomycin, enomycin, auristatin and the like. Cytotoxicagents also include radiochemicals made by conjugating radioisotopes toantibodies raised against prostate cancer proteins, or binding of aradionuclide to a chelating agent that has been covalently attached tothe antibody. Targeting the therapeutic moiety to transmembrane prostatecancer proteins not only serves to increase the local concentration oftherapeutic moiety in the prostate cancer afflicted area, but alsoserves to reduce deleterious side effects that may be associated withthe therapeutic moiety.

Binding Affinity of Antibodies of the Invention

[0084] Binding affinity for a target antigen is typically measured ordetermined by standard antibody-antigen assays, such as Biacorecompetitive assays, saturation assays, or immunoassays such as ELISA orRIA.

[0085] Such assays can be used to determine the dissociation constant ofthe antibody. The phrase “dissociation constant” refers to the affinityof an antibody for an antigen. Specificity of binding between anantibody and an antigen exists if the dissociation constant (K_(D)=1/K,where K is the affinity constant) of the antibody is <1 μM, preferably<100 nM, and most preferably <0.1 nM. Antibody molecules will typicallyhave a K_(D) in the lower ranges. K_(D)=[Ab-Ag]/[Ab][Ag] where [Ab] isthe concentration at equilibrium of the antibody, [Ag] is theconcentration at equilibrium of the antigen and [Ab-Ag] is theconcentration at equilibrium of the antibody-antigen complex. Typically,the binding interactions between antigen and antibody include reversiblenoncovalent associations such as electrostatic attraction, Van der Waalsforces and hydrogen bonds.

[0086] The antibodies of the invention specifically bind to TMEFF2proteins. By “specifically bind” herein is meant that the antibodiesbind to the protein with a K_(D) of at least about 0.1 mM, more usuallyat least about 1 μM, preferably at least about 0.1 μM or better, andmost preferably, 0.01 μM or better.

Immunoassays

[0087] The antibodies of the invention can be used to detect TMEFF2 orTMEFF2 expressing cells using any of a number of well recognizedimmunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241;4,376,110; 4,517,288; and 4,837,168). For a review of the generalimmunoassays, see also Asai (ed. 1993) Methods in Cell Biology Vol. 37,Academic Press, New York; Stites & Terr (eds. 1991) Basic and ClinicalImmunology 7th Ed.

[0088] Thus, the present invention provides methods of detecting cellsthat express TMEFF2. In one method, a biopsy is performed on the subjectand the collected tissue is tested in vitro. The tissue or cells fromthe tissue is then contacted, with an anti-TMEFF2 antibody of theinvention. Any immune complexes which result indicate the presence of aTMEFF2 protein in the biopsied sample. To facilitate such detection, theantibody can be radiolabeled or coupled to an effector molecule which isa detectable label, such as a radiolabel. In another method, the cellscan be detected in vivo using typical imaging systems. Then, thelocalization of the label is determined by any of the known methods fordetecting the label. A conventional method for visualizing diagnosticimaging can be used. For example, paramagnetic isotopes can be used forMRI. Internalization of the antibody may be important to extend the lifewithin the organism beyond that provided by extracellular binding, whichwill be susceptible to clearance by the extracellular enzymaticenvironment coupled with circulatory clearance.

[0089] TMEFF2 proteins can also be detected using standard immunoassaymethods and the antibodies of the invention. Standard methods include,for example, radioimmunoassay, sandwich immunoassays (including ELISA),immunofluorescence assays, Western blot, affinity chromatography(affinity ligand bound to a solid phase), and in situ detection withlabeled antibodies.

Administration of Pharmaceutical and Vaccine Compositions

[0090] The antibodies of the invention can be formulated inpharmaceutical compositions. Thus, the invention also provide methodsand compositions for administering a therapeutically effective dose ofan anti-TMEFF2 antibody. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. See, e.g., Ansel, et al. (1999) Pharmaceutical DosageForms and Drug Delivery; Lieberman (1992) Pharmaceutical Dosage Forms(vols. 1-3), Dekker, ISBN 0824770846, 082476918X, 0824712692,0824716981; Lloyd (1999) The Art Science and Technology ofPharmaceutical Compounding Amer. Pharm. Assn.; and Pickar (1999) DosageCalculations Thomson. Adjustments for cancer degradation, systemicversus localized delivery, and rate of new protein synthesis, as well asthe age, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art. U.S. Ser. No. 09/687,576 further discloses the use ofcompositions and methods of diagnosis and treatment in prostate canceris hereby expressly incorporated by reference.

[0091] A “patient” for the purposes of the present invention includesboth humans and other animals, particularly mammals. Thus the methodsare applicable to both human therapy and veterinary applications. In thepreferred embodiment the patient is a mammal, preferably a primate, andin the most preferred embodiment the patient is human.

[0092] The administration of the antibodies of the present invention canbe done in a variety of ways as discussed above, including, but notlimited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly.

[0093] The pharmaceutical compositions of the present invention comprisean antibody of the invention in a form suitable for administration to apatient. In the preferred embodiment, the pharmaceutical compositionsare in a water soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

[0094] The pharmaceutical compositions may also include one or more ofthe following: carrier proteins such as serum albumin; buffers; fillerssuch as microcrystalline cellulose, lactose, corn and other starches;binding agents; sweeteners and other flavoring agents; coloring agents;and polyethylene glycol.

[0095] The pharmaceutical compositions can be administered in a varietyof unit dosage forms depending upon the method of administration. Forexample, unit dosage forms suitable for oral administration include, butare not limited to, powder, tablets, pills, capsules and lozenges. It isrecognized that antibodies when administered orally, should be protectedfrom digestion. This is typically accomplished either by complexing themolecules with a composition to render them resistant to acidic andenzymatic hydrolysis, or by packaging the molecules in an appropriatelyresistant carrier, such as a liposome or a protection barrier. Means ofprotecting agents from digestion are well known in the art.

[0096] The compositions for administration will commonly comprise anantibody of the invention dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter. These compositions maybe sterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, e.g., sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of active agentin these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thepatient's needs (e.g., (1980) Remington's Pharmaceutical Science (18thed.); and Hardman, et al. (eds. 2001) Goodman & Gilman: ThePharmacological Basis of Therapeutics).

[0097] Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages are possible in topicaladministration. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art,e.g., Remington's Pharmaceutical Science and Goodman and Gilman: ThePharmacological Basis of Therapeutics, supra.

[0098] The compositions containing antibodies of the invention can beadministered for therapeutic or prophylactic treatments. In therapeuticapplications, compositions are administered to a patient suffering froma disease (e.g., a cancer) in an amount sufficient to cure or at leastpartially arrest the disease and its complications. An amount adequateto accomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health. Single ormultiple administrations of the-compositions may be administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the agents of this invention to effectively treat thepatient. An amount of modulator that is capable of preventing or slowingthe development of cancer in a mammal is referred to as a“prophylactically effective dose.” The particular dose required for aprophylactic treatment will depend upon the medical condition andhistory of the mammal, the particular cancer being prevented, as well asother factors such as age, weight, gender, administration route,efficiency, etc. Such prophylactic treatments may be used, e.g., in amammal who has previously had cancer to prevent a recurrence of thecancer, or in a mammal who is suspected of having a significantlikelihood of developing cancer.

[0099] It will be appreciated that the present prostate cancerprotein-modulating compounds can be administered alone or in combinationwith additional prostate cancer modulating compounds or with othertherapeutic agent, e.g., other anti-cancer agents or treatments.

[0100] In some embodiments, the antibodies of the invention can be usedto prepare targeted liposomes for delivery of a desired therapeuticcomposition (e.g., anti-cancer agents) to a target cell (e.g., aprostate cancer cell). The preparation and use of immunoliposomes fortargeted delivery of antitumor drugs is reviewed in Mastrobattista, etal. (1999) Advanced Drug Delivery Reviews 40:103-127.

[0101] Liposomes are vesicular structures based on lipid bilayers. Theycan be as small as 20 nm and as large as 10 μm in diameter. They can beunilamellar (only one bilayer surrounds an aqueous core) ormultilamellar (several bilayers concentrically oriented around anaqueous core). The liposomes of the present invention are formed fromstandard vesicle-forming lipids, which generally include neutral andnegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally guided by consideration of, e.g.,liposome size and stability of the liposomes in the bloodstream.

[0102] Targeting of liposomes using a variety of targeting agents (e.g.,monoclonal antibodies of the invention) is well known in the art. See,e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044). Standard methods forcoupling targeting agents to liposomes can be used. Antibody targetedliposomes can be constructed using, for instance, liposomes whichincorporate protein A. See, Renneisen, et al. (1990) J. Biol. Chem.265:16337-16342; and Leonetti, et al. (1990) Proc. Nati. Acad. Sci. USA87:2448-2451.

[0103] A variety of methods are available for preparing liposomes, asdescribed in, e.g., Szoka, et al. (1980) Ann. Rev. Biophys. Bioeng.9:467; U.S. Pat. Nos. 4, 235,871; 4,501,728; and 4,837,028. One methodproduces multilamellar vesicles of heterogeneous sizes. In this method,the vesicle forming lipids are dissolved in a suitable organic solventor solvent system and dried under vacuum or an inert gas to form a thinlipid film. If desired, the film may be redissolved in a suitablesolvent, such as tertiary butanol, and then lyophilized to form a morehomogeneous lipid mixture which is in a more easily hydrated powder-likeform. This film is covered with an aqueous solution of the targeted drugand the targeting component (antibody) and allowed to hydrate, typicallyover a 15-60 minute period with agitation. The size distribution of theresulting multilamellar vesicles can be shifted toward smaller sizes byhydrating the lipids under more vigorous agitation conditions or byadding solubilizing detergents such as deoxycholate.

Kits for Use in Diagnostic and/or Prognostic Applications

[0104] For use in diagnostic, research, and therapeutic applicationssuggested above, kits are also provided by the invention. In thediagnostic and research applications such kits may include any or all ofthe following: assay reagents, buffers, and TMEFF2-specific antibodiesof the invention. A therapeutic product may include sterile saline oranother pharmaceutically acceptable emulsion and suspension base.

[0105] In addition, the kits may include instructional materialscontaining directions (e.g., protocols) for the practice of the methodsof this invention. While the instructional materials typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES Example 1

[0106] Approximately 12 anti-TMEFF2 hybridoma supernatants were selectedfrom an initial pool of roughly one hundred, based on off rates (kd) forbinding to covalently immobilized TMEFF2-FLAG protein as measured byBIAcore™. Supernatants exhibiting the lowest dissociation rate constantswere chosen for larger scale purification. The sequences of variableregions of antibodies TMEFF2 #19, TMEFF2 #10, TMEFF2 #18, TMEFF2 #20,TMEFF2 #21 are presented in Table 1. A kinetic evaluation was carriedout on each purified antibody by measuring binding to TMEFF2-FLAG over arange of antigen concentrations. Affinity constants (K_(D)) were thendetermined using the global fitting procedure described in theBIAapplications Handbook Biacore AB, BIAapplications Handbook, versionAB, 1998, Application Notes, Note 101 (June 1995); Daiss, et al. (1994)Methods: A companion to Methods in Enzymology Volume 6, p143-156. Inaddition, pair-wise epitope mapping was carried out through acompetitive binding analysis. This was accomplished by exposing theTMEFF2-FLAG surface to a saturating amount of one antibody sample andmeasuring the response level of a second injected antibody. Using thismethodology antibodies recognizing a number of individual epitopes wereselected for further study.

[0107] Each antibody of interest was covalently coupled to the synthetictoxin auristatin (Int. J. Oncol. 15:367-72 (1999)) (pAE), a dolastatin10 derivative, and assessed for TMEFF2 dependent cell death in vitro.The cell death assay (Proc. Nat'l Acad. Sci. USA 93:8618-23(1996)) wasexecuted by first determining a cell density that exhibits linear cellgrowth over several days. Populations of dividing cells were thenincubated with multiple concentrations of toxin-conjugated TMEFF2antibodies (or a negative control) for one hour, followed by removal ofthe antibody and gentle washing. Four days later, cell viability wasdetermined by using the Celltiter 96 assay (Promega). In this manner aprostate cancer cell line stably expressing TMEFF2 (PC3-TMEFF2), wascompared with the parental cell line that does not (PC3).

[0108] Two antibodies corresponding to distinct epitopes, as determinedby BIAcore, have been assessed for their ability to interfere with cellsurvival in vitro. One of these antibodies, TMEFF2 #19-pAE, appears topromote significant cell death in PC3-TMEFF2 cells, but not in theparental line. The other antibody, #21-pAE, also causes cell death, butwith somewhat less potency than #19-pAE. A negative control antibodythat does not recognize a cell surface marker in PC3 cells, TIB-pAE,does not affect cell survival in either cell line. Additionally, anotherprostate cancer line, LnCAP, which has been determined to express smallamounts of surface TMEFF2, also displayed sensitivity to #1 9-pAErelative to TIB-pAE. These results show that #19-pAE is a potent andselective cytotoxic agent on TMEFF2 expressing cells. TABLE 1TMEFF2#19.Heavy chain variable region. SEQ ID NO: 1GATGTACAACTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAGCTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTTCATAAGCTACGACGGTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTGAGAACCAGTTTTTCCTGAACTTGAGATCTGTGACTACTGAGGACACAGCAACATATTATTGTGCAAGAGGTTTACGACGAGGGGACTATTCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO:2DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWSWIRQFPGNKLEWMGFISYDGSNKYNPSLKNRISITRDTSENQFFLNLRSVTTEDTATYYCARGLRRGDYSMDYWGQGTSVTVSS TMEFF2#19.Light chainvariable region SEQ ID NO: 3GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGTGTCAGCATCACCTGCAAGGCCAGTCAGAATGTCGTTACAGCTGTAGCCTGGTATCGACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCAATCGGCACACTGGAGTCCCTGACCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAATATGCAGTCTGAAGACCTGGCAGATTATTTCTGCCAGCAATATAGCAGCTATCCGTTCACGTTCGGACGGGGGACCAAGCTGGAAATAAAA SEQ ID NO: 4DIVMTQSQKFMSTSVGDSVSITCKASQNVVTAVAWYRQKPGQSPKLLIYSASNRHTGVPDRFTGSGSGTDFTLTINNMQSEDLADYFCQQYSSYPFTFGGGTKLEIK TMEFF2#10. heavy chain variable regionSEQ ID NO: 5GAAGTGAACCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTTCTGGATTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGGTAATACCTATTATTCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTCCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACGGGGATTACGACGAGGGGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO:6EVNLVESGGGLVQPGGSLKLSCATSGFTFSDYYMFWIRQTPEKRLEWVAYISNGGGNTYYSDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLRRGGAMDYWGQGTSVTVSS TMEFF2#10. Light chainvariable region SEQ ID NO: 7GACATTGTTTTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTACGGTGGTTATGGTTATATAAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGATTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAGTCTATTACTGTCAACAAAGTTATGTGGATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAATC SEQ ID NO: 8DIVLTQSPASLAVSLGQRATISCKASQSVDYGGYGYINWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAAVYYCQQSYVDPFTFGSGTKLEII TMEFF2#18. Heavy Chain variableregion SEQ ID NO: 9CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAGCTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGGCGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGGGGGTGATGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 10QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMSWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCGGDAYWGQGTLVTVSA TMEFF2#18. Light Chain variableregion SEQ ID NO: 11GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCACCACAGTAGGGAGCTTCGGACGTTCGGTGGAGGCACCAAACTGGAAATCAAA SEQ ID NO: 12DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELRTFGGGTKLEIK TMEFF2#20. Heavy Chain variableregion SEQ ID NO: 13GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGATGAAGCCTGGGGCTTCAGTGAAGATATCTTGCAAGGCTTCTACTTACTCATTCACTAGGTACTTCATGCACTGGGTGAAGCAGAGCCATGGAGAGAGCCTTGAGTGGATTGGATATATTGATCCTTTCAATGGTGGTACTGGCTACAATCAGAAATTCAAGGGCAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCTACATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAACGTATGGCTCCGACTACTTTCACTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO: 14EIQLQQSGPELMKPGASVKISCKASTYSFTRYFMHWVKQSHGESLEWIGYIDPFNGGTGYNQKFKGKATLTVDKSSSTAYMHLSSLTSEDSAVYYCVTYGSDYFDYWGQGTTLTVSS TMEFF2#20. Light chainvariable region SEQ ID NO: 15GACATTGTGATGACCCAGCCACAAAAATTCATGTCCACGTCTGTAGGCGACAGGGTCAGTGTCACCTGCAAGGCCAGTCAGAATGTGGAAACTGATGTAGTCTGGTATCAACAGAAACCTGGGCAACCACCTAAAGCACTGATTTACTCGGCATCCTACCGGCACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAAATTTCACTCTCACCATCAGCACTGTACAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAACTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAATA SEQ ID NO: 16DIVMTQPQKFMSTSVGDRVSVTCKASQNVETDVVWYQQKPGQPPKALIYSASYRHSGVPDRFTGSGSGTNFTLTISTVQSEDLAEYFCQQYNNYPFTFGSGTKLEII TMEFF2#21. Heavy chain variable regionSEQ ID NO: 17CAGATCCACTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACAAACTTTGCAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTCAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGTCAGTATTGCCTATTTGCAGATCAACAGCCTCAAAAATGAGGACACGGCTACATATTTCTGTTCAAAATTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO: 18QIHLVQSGPELKKPGETVKISCKASGYTFTNFAMNWVKQAPGKGFKWMGWINTYTGEPTYADDFKGRFAFSLETSVSIAYLQINSLKNEDTATYPCSKFDYWGQGTTLTVSS TMEFF2#21 .Light Chain variableregion SEQ ID NO: 19GACATCCAGATGACTCAGTCTCCAGCCTCCCTATATGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTTTCAGCAGAAACAGGGAAAATCTCCTCACCTCCTGGTCTATAATGCAAAAACCTTAGCAGCAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCACCAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCCACGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA SEQ ID NO: 20DIQMTQSPASLYASVGETVTITCRASENIYSYLAWFQQKQGKSPHLLVYNAKTLAAGVPSRFSGSGSGTQFSLKITSLQPEDFGSYYCQHHYGTPTWTFGGGTKLEIK

[0109] Relatively low amounts of the TMEFF2 protein are detectable onthe cell surface of cancer cell lines, as assessed by FACS analysisusing the TMEFF2 #19 antibody. Thus, the effectiveness of thetoxin-conjugated #19 antibody at killing cells specifically expressingthis target was surprising. However, experiments designed to assess theability of specific antibody:target combinations to be internalized hasgenerated novel data that explains the efficiency of thetoxin-conjugated anti TMEFF2 antibodies at killing. It has becomeapparent that this particular target protein shows an incredibly highrate of internalization. In these internalization experiments, cellsexpressing TMEFF2 are incubated at different temperatures, and fordifferent lengths of time, in the presence of anti-TMEFF2 antibody.After incubation with anti- TMEFF2 antibody for 1 hour at 4° C., thecells are washed and further incubated with a fluorescently labeledanti-mouse antibody. By fluorescent microscopy a low level of specificantibody binding to the TMEFF2 at the cell surface is observed. Incontrast, when cells are incubated at 37° C. for 1 hour, a temperaturethat allows for protein trafficking and internalization, and are thensubjected to permeabilization and staining with the fluorescentlylabeled anti-mouse antibody, the majority of the fluorescence isdetected within the cells. Such data indicates that the specificantibody:target combination has been internalized—a result that isfurther confirmed by subjecting the cells to an acid stripping stepprior to the detection step. The acid stripping removes all proteinstill present at the cell surface leaving behind only the internalizedantibody:target proteins. In contrast to other antibody:targetcombinations such as herceptin:Her2 and anti-ephrinA3: ephrinA3, theseexperiments have shown that the TMEFF2 protein, as recognized by thespecific anti-TMEFF2 antibodies, is internalized at a very rapid rateand also that almost complete internalization of the cell surfaceprotein is observed within the 1 hour period. These data, showing thesurprisingly efficient internalization of TMEFF2 account for theefficiency of the toxin-conjugated anti-TMEFF2 antibodies at killing.

Example 2

[0110] Using standard techniques as described above, humanized TMEFF2#19antibodies were generated. The sequences of four humanized heavy chainvariable regions and three humanized light chain variable regions arepresented in Table 2. The heavy and light chain variable regions may beused to combine into binding sites, and among the tested combinations,retain binding affinity. These antibodies can be used in in vivo mousemodels to inhibit growth of tumor cells in vivo. TABLE 2 VH 1.0 DNA SEQID NO: 21GATGTACAACTTCAGGAGTCAGGACCTGGCCTCOTCAAACCTTCTGAGACCCTGTCTCTCACCTGCGCAGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAGCTGGATCCGGCAGTTTCCAGGAAAGAAACTGGAATGGATCCGCTTCATAACCTACGACCCTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTGAGAACCAGTTTTTCCTGAACTTGTCTTCTGTCACTCCAGCACACACAGCAACATATTATTGTGCAAGAGGTTTACGACGAGGGCACTATTCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCA VH 1.0AMINO ACIDS SEQ ID NO: 22DVQLQESGPGLVKPSETLSLTCAVTGYSITSGYYWSWIRQFPGKKLEWMGFISYDGSNKYNPSLKNRISITRDTSENQFFLKLSSVTAADTATYYCARGLRRGDYSMDYWGQGTLVTVSS VH 2.0 DNA SEQ ID NO: 23GATGTACAACTTCAGGACTCAGGACCTGGCCTCCTGAAACCTTCTGAGACCCTGTCTCTCACCTGCGCAGTCACTCGCTACTCCATCACCAGTGGTTATTACTGGAGCTGGATCCGGCAGCCTCCACGAAAGGGCCTGCAATGGATGCGCTTCATAAGCTACGACGGTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTGAGAACCAGTTTTTCCTCAAGTTGTCTTCTGTGACTGCACCAGACACACCAGTCTATTATTGTGCAAGAGGTTTACGACGAGGCCACTATTCTATGCACTACTGGGGTCAAGGAACCCTCGTCACCGTCTCCTCA VH 2.0AMINO ACIDS SEQ ID NO: 24DVQLQESGPGLVKPSETLSLTCAVTGYSITSGYYWSWIRQPPGKGLEWMGFISYDGSNKYNPSLKNRISITRDTSENQFFLKLSSVTAADTAVYYCARGLRRGDYSMDYWGQGTLVTVSS VH 3.0 DNA SEQ ID NO: 25GATGTACAACTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTGAGACCCTGTCTCTCACCTCCGCAGTCAGCGGCTACTCCATCACCAGTGGTTATTACTGGAGCTGGATCCGGCAGCCTCCAGGAAAGGGCCTCCAATGGATGGGCTTCATAAGCTACGACGGTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCACCATCTCCCGTGACACATCTAAGAACCAGTTTTCCCTGAAGTTGTCTTCTGTCACTGCAGCACACACACCAGTCTATTATTGTGCAACAGGTTTACGACGAGGGCACTATTCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCA VH 3.0AMINO ACIDS SEQ ID NO: 26DVQLQESGPGLVKPSETLSLTCAVSGYSITSGYYWSWIRQPPGKGLEWMGFISYDGSNKYNPSLKNRITISRDTSKNQFSLKLSSVTAADTAVYYCARGLRRGDYSMDYWGQGTLVTVSS VH 4.0 DNA SEQ ID NO: 27GATGTACAACTTCAGGAGTCAGGACCTGCCCTCGTGAAACCTTCTCACACCCTGTCTCTCACCTGCGCAGTCAGCGGCTACTCCATCACCAGTGCTTATTACTGGAGCTCCATCCGGCAGTTTCCAGGAAAGAPACTGGAATCGATGGGCTTCATAAGCTACCACGGTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCACCATCTCCCGTGACACATCTAAGAACCAGTTTTCCCTGAAGTTGTCTTCTGTGACTGCAGCAGACACAGCAACATATTATTGTGCAAGAGGTTTACGACGAGGGCACTATTCTATCGACTACTCGCGTCAAGGAACCCTGGTCACCCTCTCCTCA VH 4.0AMINO ACIDS SEQ ID NO: 20DVQLQESGPGLVKPSETLSLTCAVSGYSITSGYYWSWIRQFPGKKLEWMGFISYDGSNKYNPSLKNRITISRDTSKNQFSLKLSSVTAADTATYYCARGLRRGDYSMDYWGQGTLVTVSS VL 1.0 DNA SEQ ID NO: 29GACATTCAGATGACCCAGTCTCAATCTAGTATGTCCACATCAGTACGAGACCGAGTCACCATCACCTGCAAGGCCAGTCAGAATGTCGTTACAGCTGTACCCTGGTATCGACAGAAACCAGGAAAGTCTCCTAAACTACTGATTTACTCGGCATCCAATCGCCACACTGGAGTCCCTAGTCGCTTCTCTGCCAGTGCATCTCCCACAGATTTCACTCTCACCATCTCTAGCATGCAGCCTGAAGACTTCGCAGATTATTTCTGCCAGCAATATACCAGCTATCCGTTCACGTTCGGACGGCGGACCAAGCTCCAGATCAAACGC VL 1.0 AMINO ACIDS SEQ ID NO: 30IQMTQSQSSMSTSVGDRVTITCKASQNVVTAVAWYRQKPGKSPKLLIYSASNRHTGVPSRFSGSGSGTDFTLTISSMQPEDFADYFCQQYSSYPFTFGGGTKLEIKR VL 2.0 DNA SEQ ID NO: 31GACATTCAGATGACCCAGTCTCCATCTAGTCTGTCCCCTTCAGTACGAGACCGAGTCACCATCACCTGCAAGGCCAGTCACAATGTCGTTACAGCTGTAGCCTGGTATCGACACAAACCAGGAAAGTCTCCTAAACTACTGATTTACTCCGCATCCAATCCGCACACTGCAGTCCCTAGTCCCTTCTCTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCTCTAGCCTGCAGCCTGAAGACTTCGCAGATTATTTCTGCCAGCAATATAGCAGCTATCCGTTCACGTTCGCACGGGGGACCAAGGTCGAGATCAAACGG VL 2.0 AMINO ACIDS SEQ ID NO: 32DIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYRQKPGKSPKLLIYSASNRHTGVPSRFSGSGSGTDFTLTISSLQPEDFADYFCQQYSSYPFTFGGGTKVEIKR VL 3.0 DNA SEQ ID NO: 33GACATTCAGATGACCCAGTCTCCATCTAGTCTGTCCGCTTCAGTAGGAGACCGAGTCACCATCACCTGCAAGGCCAGTCACAATCTCGTTACAGCTGTAGCCTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAACTACTGATTTACTCCGCATCCAATCGGCACACTGGAGTCCCTAGTCGCTTCTCTGGCAGTGGATCTCGCACAGATTTCACTCTCACCATCTCTACCCTCCAGCCTGAACACTTCGCAACCTATTATTGCCAGCAATATAGCACCTATCCGTTCACGTTCCGACGGGGCACCAACGTCGAGATCAAACGC VL 3.0 AMINO ACIDS SEQ ID NO: 34DIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKAPKLLIYSASNRHTCVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPFTFGGGTKVEIKR

Example 3 Auristatin E Conjugated Anti-TMEFF2 Antibodies Target and KillProstate Cancer Tumors in Vivo

[0111] The TMEFF2 gene is highly and specifically expressed in clinicalprostate cancer samples. To demonstrate that the protein product of theTMEFF2 gene is a therapeutic target for the treatment of prostatecancer, the human prostate cancer cell line LNCAP was modeled in SCID(severe combined immunodeficient) mice. Gene expression analysis showsthat TMEFF2 is highly expressed in LNCAP cells grown on plastic intissue culture and also when grown as xenograft tumors in SCID mice.

[0112] To determine the in vivo effects of toxin-conjugated anti-TMEFF2antibodies (#19-pMMVCAE), LNCAP cells were grown as xenograft tumors inSCID mice. After the tumors reached a certain size (average of 100 mm³),the animals were distributed into 3 groups and subjected to treatmentwith either a) control vehicle, b) #19-pMMVCAE, or c) isotypecontrol-MMVCAE (an antibody that does not recognize molecules on thesurface of LNCAP cells). Conjugated antibodies were used at 0.25 mg/kgof drug equivalent (˜5 mg/kg of antibody-drug conjugate), and wereadministered at 4 day intervals. Tumor size was measured twice a week.Animal weight was monitored throughout the experiment and serum PSA(prostate-specific antigen) levels were measured at various timeintervals during the experiment.

[0113] The results showed that treatment with #19-pMMVCAE significantlyreduced LNCAP tumor growth. In fact, established LNCAP tumors regressedin size (to less than 100 mm³), serum PSA (a surrogate marker forprostate tumor burden) levels significantly dropped (<10 ng/ml), whileanimal weight remained steady and animals appeared healthy. This is incontrast to mice that received either control vehicle or the isotypecontrol-MMVCAE. The tumors in these mice grew rapidly and had to besacrificed at days 50-60 post tumor implantation due to the large sizeof the tumors (>500 mm³). In addition, the animals lost considerableamount of weight, appeared moribund and had significantly higher levelsof serum PSA (>350 ng/ml). Treatment with humanized #19-pMMVCAE (seeExample 2) of mice bearing LNCAP tumors elicited similar results as seenwith the murine antibody, e.g., established tumors regressed, serum PSAlevels dropped and animals appeared healthy.

[0114] These results indicate that TMEFF2 protein is a new therapeutictarget for the treatment of prostate cancer and other prostatic diseases(such as benign prostatic hyperplasia-BPH) that exhibit TMEFF2expression. In fact, anti-TMEFF2 treatment will allow for a moreeffective treatment of prostate cancer and BPH patients while reducingthe need for surgery, radiation and chemotherapeutic treatment.

Example 4 Immunohistochemical Analysis of TMEFF2 in Clinical SamplesShows Significant Protein Expression in Prostate Cancer

[0115] To determine how prevalent the TMEFF2 protein target is inprostate cancer patients, immunohistochemistry (IHC) was performed onclinical specimens derived from radical prostatectomies of patients thatexhibited localized prostate cancer (Gleason grades 3-5). In addition, asmall number of lymph node metastases of prostate cancer and advanced D2stage prostate cancer samples were analyzed.

[0116] To perform IHC on these clinical specimens, a monoclonal antibodydirected to TMEFF2 (clone #19) was used on tissue microarrays andindividual slides of prostate cancer specimens. Tissue microarrays weregenerated by incorporating tissue core biopsies of 1.0 mm intomedium-density tissue microarrays (Beecher Instruments, Silver Spring,Md.) employing the technique described by Kononen, et al. (1998) NatureMed 4:844-847). Hematoxylin and eosin stained template sections of theradical prostatectomy paraffin donor blocks were marked up for areas ofnodular hyperplasia and cancer by a histopathologist. Using thesesections as a guide, 1-2 cores of nodular hyperplasia adjacent to cancer(≦2 cm from the cancer) and 2-4 cores of cancer were sampled from theparaffin donor blocks of each of the radical prostatectomy specimens andincorporated directly into recipient array blocks. A core was includedfrom each of the primary, secondary and tertiary Gleason patternsrepresented in the cancers. The normal prostate, lymph node metastasesspecimen, and D2 stage specimens were mounted as conventional tissuesections.

[0117] Immunohistochemical (IHC) staining for TMEFF2 was performed onroutinely processed, paraffin-embedded tissue specimens. Four μmsections of these specimens were cut, mounted on Superfrost Plusadhesion slides (Lomb Scientific, Sydney, Australia), and heated in aconvection oven at 75° C. for 2 hours to promote adherence to the slide.Paraffin-embedded pellets of LNCAP and PC-3 prostate cancer cell lineswere used as positive and negative controls, respectively. Sections werede-waxed and rehydrated before unmasking in EDTA/Citrate buffer and werethen stained with anti-TMEFF2 antibody. Anti-TMEFF2 signal was detectedusing DAKO EnVision Plus Labeled Polymer (DAKO Corporation, Carpinteria,Calif.) with liquid 3,3′-diaminobenzidine Plus (DAKO Corporation,Carpinteria, Calif.) as substrate. Counterstaining was performed withhematoxylin and Scott's blueing solution. All TMEFF2 immunostaining wascytoplasmic and the intensity of staining was graded on the density ofcytoplasmic granules as negative, weak, moderate, or strong.

[0118] The results show that anti-TMEFF2 staining was restrictedexclusively to the cytoplasm and membranes of prostatic epithelial cellswith no nuclear or stromal staining. Benign prostatic tissue displayedsome TMEFF2 protein expression with weak to moderate staining seen innormal prostate specimens and weak to moderate staining seen in theBenign Prostatic Hyperplasia (BPH samples). Expression in areas ofhyperplasia adjacent to cancer also showed moderate staining in most ofthe cases examined. The prostate cancer cohort (n=241) displayed weak tostrong staining in 176 cases, demonstrating that a large fraction ofprostate cancer patients exhibit expression of TMEFF2. TMEFF2-positivitywas also detected in 4/6 cases of locally advanced disease (D2 stage)and 3/5 lymph node metastatic lesions, indicating that expression ofthis target is retained in advanced stage disease.

[0119] Intensity of immunostaining for TMEFF2 protein in normalnon-prostate body tissues was consistent with the levels of RNAexpression detected in the transcript profiling. Only brain showed lowlevels of TMEFF2 expression. No expression was detected in the followingnormal tissues: bladder, cervix, small intestine, spinal cord,myometrium, pancreas, skin, colon, liver, heart, kidney, testes, lung,adrenal gland, skeletal muscle, spleen, and lymph node. This dataconfirms the prostate and prostate cancer specificity of TMEFF2.

[0120] These results, combined with the antibody-drug conjugate mediatedkilling of TMEFF2 expressing tumor cells, indicate that TMEFF2 is a goodtherapeutic target for the treatment of prostate cancer.

Example 5 Use of TMEFF2 Antibodies to Delay the Onset ofAndrogen-independence of Prostate Cancer and/or to TreatAndrogen-Independent Disease

[0121] Prostate cancer is a hormone regulated disease that affects menin the later years of life. Untreated prostate cancer metastasizes tolymph nodes and bone in advanced cases. In such cases current treatmentconsists of antagonizing the androgenic growth-stimulus that feeds thetumor by chemical or surgical hormone-ablation therapy (Galbraith andDuchesne. (1997) Eur. J. Cancer 33:545-554). An unfortunate consequenceof anti-androgen treatment is the development of androgen-independentcancer. Androgen regulated genes, such as the gene encodingprostate-specific antigen (PSA), are turned off with hormone-ablationtherapy, but reappear when the tumor becomes androgen-independent(Akakura et al. (1993) Cancer 71:2782-2790).

[0122] To study the progression of androgen-dependent prostate cancer toandrogen-independent prostate cancer the human CWR22 prostate cancerxenograft model was propagated in nude mice (see Pretlow, et al. (1993)J. Natl. Cancer Inst. 85:394-398). The CWR22 xenograft isandrogen-dependent when grown in male Nude mice. Androgen-independentsub-lines can be derived by first establishing androgen-dependent tumorsin male mice. The mice are then castrated to remove the primary sourceof growth stimulus (androgen), resulting in tumor regression. Within 3-4months molecular events prompt the tumors to relapse and start growingas androgen-independent tumors. See, e.g., Nagabhushan, et al. (1996)Cancer Res. 56:3042-3046; Amler, et al. (2000) Cancer Res. 60:6134-6141;and Bubendorf, et al. (1999) J. Natl. Cancer Inst. 91:1758-1764.

[0123] Using the CWR22 xenograft model we have previously monitored thegene expression changes that occur during the transition fromandrogen-dependence to androgen-independence (see WO02098358). Tumorswere grown subcutaneously in male nude mice. Tumors were harvested atdifferent times after castration. The time points ranged from 0 to 125days post-castration. Castration resulted in tumor regression. At day120 and thereafter, the tumors relapsed and started growing in theabsence of androgen.

[0124] Gene expression profiling of the harvested tumors wasaccomplished using the Eos Hu03 oligonucleotide microarray (AffymetrixEos Hu03). Our results identified several hundred genes that exhibitedsignificant gene expression changes associated with androgen ablationtherapy. Some genes were associated with the androgen-dependent growthphase of the CWR22 tumors (pre-castration and 1-5 days post-castration),some genes were associated with the androgen-withdrawal phase (10-82days post castration, characterized by tumor regression and/or tumorgrowth stasis), and some genes were associated with theandrogenindependent growth of CWR22 (greater than 120 days postcastration). See WO02098358.

[0125] The gene encoding TMEFF2 showed high expression levels throughoutsuch a whole androgen-withdrawal experiment. Highest expression levelswere seen in the androgen-dependent CWR22 xenografts (confirmed byimmunohistochemistry for the presence of TMEFF2 protein) and in theemerging androgen-independent CWR22 tumors (>120 days post-castration).Lower, but still significant expression was detected in tumors 10-82days post castration (androgen-withdrawal phase).

[0126] To prevent androgen-independent prostate cancer, CWR22 tumorbearing mice are treated, post androgen-ablation therapy, withanti-TMEFF2 antibody conjugated to Auristatin E (#19-pMMVCAE). Theobjective is to show that post-castration treatment with #19-pMMVCAEduring the androgen-withdrawal phase (10-82 days post castration) willresult in a delay in the onset of androgen-independent CWR22 tumorgrowth. CWR22 tumors are grown in male immunodeficient mice for 2-3weeks. The mice are then castrated to induce tumor regression and entryinto the androgen-withdrawal phase. Twenty days post-castration thetumors are treated with #19-pMMVCAE as described in Example 3. Asignificant effect of #1 9-pMMVCAE would manifest itself in a delay inthe onset of androgen-independence (e.g., 5 months or more postcastration). This would suggest that patients with advanced stageprostate cancer, that are treated with androgen-ablation therapy, wouldgreatly benefit from treatment with humanized #1 9-pMMVCAE. Thesepatients would at the very least enjoy a longer survival period postandrogen-ablation therapy and would possibly be cured of prostatecancer.

[0127] A non-significant effect in #I9-pMMVCAE treatment may be due toseveral potential factors: (a) CWR22 xenograft tumors may be resistantto Auristatin E; (b) the tumor cells may not efficiently internalize#19-pMMVCAE during the androgen-withdrawal phase; or (c) TMEFF2 proteinexpression may be significantly decreased during the androgen-withdrawalphase. Modifications in treatment are available to address these issues.

[0128] To treat androgen-independent prostate cancer, CWR22 tumorbearing mice are treated at the time of onset of androgen-independencewith #19-pMMVCAE. The objective is to show that post-castrationtreatment with #1 9-pMMVCAE during the emergence ofandrogen-independence (>120 days post castration) will result inregression of androgen-independent CWR22 tumors. CWR22 tumors are grownin male immunodeficient mice for 2-3 weeks. The mice are then castratedto induce tumor regression and entry into the androgen-withdrawal phase.Ten days after the tumors start growing in an androgen-independentmanner, the tumors are treated with #1 9-pMMVCAE as described in Example3. A significant effect of #1 9-pMMVCAE would manifest itself inregression of androgen-independent tumors. This would suggest thatpatients that were treated with androgen-ablation therapy and thatsuffered relapse in the form of androgen-independent tumor growth andmetastasis would greatly benefit from treatment with humanized #19-pMMVCAE treatment. These patients, which currently do not have analternative treatment, would at the very least enjoy a longer survivalperiod after the emergence of androgen-independent prostate cancer andwould possibly be cured of the disease.

[0129] It is understood that the examples described above in no wayserve to limit the true scope of this invention, but rather arepresented for illustrative purposes. All publications, sequences ofaccession numbers, and patent applications cited in this specificationare herein incorporated by reference as if each individual publicationor patent application were specifically and individually indicated to beincorporated by reference.

[0130] All UniGene cluster identification numbers (see,httpH//www.ncbi.nlm.nih.gov/unigene/). and accession numbers herein arefor the GenBank sequence database and the sequences of the accessionnumbers are hereby expressly incorporated by reference. GenBank is knownin the art, see, e.g., Benson, et al. (1998) Nucleic Acids Research26:1-7. Sequences are also available in other databases, e.g., EuropeanMolecular Biology Laboratory (EMBL) and DNA Database of Japan (DDBJ).

1 34 1 360 DNA Artificial Heavy chain variable region 1 gatgtacaacttcaggagtc aggacctggc ctcgtgaaac cttctcagtc tctgtctctc 60 acctgctctgtcactggcta ctccatcacc agtggttatt actggagctg gatccggcag 120 tttccaggaaacaaactgga atggatgggc ttcataagct acgacggttc caataagtat 180 aatccatctctcaaaaatcg aatctccatc actcgtgaca catctgagaa ccagtttttc 240 ctgaacttgagatctgtgac tactgaggac acagcaacat attattgtgc aagaggttta 300 cgacgaggggactattctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360 2 120 PRTArtificial Heavy chain variable region 2 Asp Val Gln Leu Gln Glu Ser GlyPro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys SerVal Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Ser Trp Ile ArgGln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Phe Ile Ser Tyr AspGly Ser Asn Lys Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile ThrArg Asp Thr Ser Glu Asn Gln Phe Phe 65 70 75 80 Leu Asn Leu Arg Ser ValThr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Arg ArgGly Asp Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val ThrVal Ser Ser 115 120 3 321 DNA Artificial Light chain variable region 3gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagtgtcagc 60atcacctgca aggccagtca gaatgtggtt acagctgtag cctggtatcg acagaaacca 120ggacaatctc ctaaactact gatttactcg gcatccaatc ggcacactgg agtccctgac 180cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcaacaa tatgcagtct 240gaagacctgg cagattattt ctgccagcaa tatagcagct atccgttcac gttcggaggg 300gggaccaagc tggaaataaa a 321 4 107 PRT Artificial Light chain variableregion 4 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly1 5 10 15 Asp Ser Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Val ThrAla 20 25 30 Val Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ser Pro Lys Leu LeuIle 35 40 45 Tyr Ser Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe ThrGly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Met GlnSer 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser TyrPro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 5360 DNA Artificial Heavy chain variable region 5 gaagtgaacc tggtggagtctgggggaggc ttagtgcagc ctggagggtc cctgaaactc 60 tcctgtgcaa cctctggattcactttcagt gactattaca tgttctggat tcgccagact 120 ccagagaaga ggctggagtgggtcgcatac attagtaatg gtggtggtaa tacctattat 180 tcagacactg taaagggccgattcaccatc tccagagaca atgccaagaa caccctgtac 240 ctccaaatga gccgtctgaagtctgaggac acagccatgt attactgtgc aagacgggga 300 ttacgacgag ggggggctatggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360 6 120 PRT ArtificialHeavy chain variable region 6 Glu Val Asn Leu Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Thr SerGly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Phe Trp Ile Arg Gln Thr ProGlu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn Gly Gly Gly AsnThr Tyr Tyr Ser Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg AspAsn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Arg Leu Lys SerGlu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Leu Arg Arg GlyGly Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val SerSer 115 120 7 333 DNA Artificial Light chain variable region 7gacattgttt tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60atctcctgca aggccagcca aagtgttgat tacggtggtt atggttatat aaactggtac 120caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa tctagaatct 180gggatcccag ccaggtttag tggcagtggg tctgggacag atttcaccct caacatccat 240cctgtggagg aggaggatgc tgcagtctat tactgtcaac aaagttatgt ggatccattc 300acgttcggct cggggacaaa gttggaaata atc 333 8 111 PRT Artificial Lightchain variable region 8 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu AlaVal Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser GlnSer Val Asp Tyr Gly 20 25 30 Gly Tyr Gly Tyr Ile Asn Trp Tyr Gln Gln LysPro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu GluSer Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Val TyrTyr Cys Gln Gln Ser Tyr 85 90 95 Val Asp Pro Phe Thr Phe Gly Ser Gly ThrLys Leu Glu Ile Ile 100 105 110 9 336 DNA Artificial Heavy chainvariable region 9 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagacagtcaagatc 60 tcctgcaagg cttctgggta taccttcaca aactatggaa tgagctgggtgaagcaggct 120 ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactggagagccaacatat 180 gctgatgact tcaaggggcg gtttgccttc tctttggaaa cctctgccagcactgcctat 240 ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgggggtgatgct 300 tactggggcc aagggactct ggtcactgtc tctgca 336 10 112 PRTArtificial Heavy chain variable region 10 Gln Ile Gln Leu Val Gln SerGly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Ser Trp Val LysGln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr TyrThr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala PheSer Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn AsnLeu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Gly Gly Asp Ala TyrTrp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 11 330 DNAArtificial Light chain variable region 11 gacattgtgc tgacacagtctcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcatgca gggccagcaaaagtgtcagt acatctggct atagttatat gcactggtac 120 caacagaaac caggacagccacccaaactc ctcatctatc ttgcatccaa cctagaatct 180 ggggtccctg ccaggttcagtggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgctgcaacctat tactgtcagc acagtaggga gcttcggacg 300 ttcggtggag gcaccaaactggaaatcaaa 330 12 110 PRT Artificial Light chain variable region 12 AspIle Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 7580 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 9095 Glu Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 11013 351 DNA Artificial Heavy chain variable region 13 gagatccagctgcagcagtc tggacctgag ctgatgaagc ctggggcttc agtgaagata 60 tcttgcaaggcttctactta ctcattcact aggtacttca tgcactgggt gaagcagagc 120 catggagagagccttgagtg gattggatat attgatcctt tcaatggtgg tactggctac 180 aatcagaaattcaagggcaa ggccacattg actgtagaca aatcttccag cacagcctac 240 atgcatctcagcagcctgac atctgaggac tctgcagtct attactgtgt aacgtatggc 300 tccgactactttgactattg gggccaaggc accactctca cagtctcctc a 351 14 117 PRT ArtificialHeavy chain variable region 14 Glu Ile Gln Leu Gln Gln Ser Gly Pro GluLeu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala SerThr Tyr Ser Phe Thr Arg Tyr 20 25 30 Phe Met His Trp Val Lys Gln Ser HisGly Glu Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Phe Asn Gly GlyThr Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val AspLys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Thr Tyr Gly Ser Asp Tyr PheAsp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 15321 DNA Artificial Light chain variable region 15 gacattgtga tgacccagccacaaaaattc atgtccacgt ctgtaggcga cagggtcagt 60 gtcacctgca aggccagtcagaatgtggaa actgatgtag tctggtatca acagaaacct 120 gggcaaccac ctaaagcactgatttactcg gcatcctacc ggcacagtgg agtccctgat 180 cgcttcacag gcagtggatctgggacaaat ttcactctca ccatcagcac tgtacagtct 240 gaagacttgg cagagtatttctgtcagcaa tataacaact atccattcac gttcggctcg 300 gggacaaagt tggaaataat a321 16 107 PRT Artificial Light chain variable region 16 Asp Ile Val MetThr Gln Pro Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg ValSer Val Thr Cys Lys Ala Ser Gln Asn Val Glu Thr Asp 20 25 30 Val Val TrpTyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Ala Leu Ile 35 40 45 Tyr Ser AlaSer Tyr Arg His Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly SerGly Thr Asn Phe Thr Leu Thr Ile Ser Thr Val Gln Ser 65 70 75 80 Glu AspLeu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Phe 85 90 95 Thr PheGly Ser Gly Thr Lys Leu Glu Ile Ile 100 105 17 336 DNA Artificial Heavychain variable region 17 cagatccact tggtgcagtc tggacctgag ctgaagaagcctggagagac agtcaagatc 60 tcctgcaagg cttctggata taccttcaca aactttgcaatgaactgggt gaagcaggct 120 ccaggaaagg gtttcaagtg gatgggctgg ataaacacctacactggaga gccaacatat 180 gctgatgact tcaagggacg gtttgccttc tctttggaaacctctgtcag tattgcctat 240 ttgcagatca acagcctcaa aaatgaggac acggctacatatttctgttc aaaatttgac 300 tactggggcc aaggcaccac tctcacagtc tcctca 336 18112 PRT Artificial Heavy chain variable region 18 Gln Ile His Leu ValGln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys IleSer Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe 20 25 30 Ala Met Asn TrpVal Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp Met 35 40 45 Gly Trp Ile AsnThr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg PheAla Phe Ser Leu Glu Thr Ser Val Ser Ile Ala Tyr 65 70 75 80 Leu Gln IleAsn Ser Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ser Lys PheAsp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 100 105 110 19 324DNA Artificial Light chain variable region 19 gacatccaga tgactcagtctccagcctcc ctatatgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtgagaatatttac agttatttag catggtttca gcagaaacag 120 ggaaaatctc ctcacctcctggtctataat gcaaaaacct tagcagcagg tgtgccatca 180 aggttcagtg gcagtggatcaggcacacag ttttctctga agatcaccag cctgcagcct 240 gaagattttg ggagttattactgtcaacat cattatggta ctcccacgtg gacgttcggt 300 ggaggcacca agctggaaatcaaa 324 20 108 PRT Artificial Light chain variable region 20 Asp IleGln Met Thr Gln Ser Pro Ala Ser Leu Tyr Ala Ser Val Gly 1 5 10 15 GluThr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 LeuAla Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro His Leu Leu Val 35 40 45 TyrAsn Ala Lys Thr Leu Ala Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 SerGly Ser Gly Thr Gln Phe Ser Leu Lys Ile Thr Ser Leu Gln Pro 65 70 75 80Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Thr 85 90 95Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 21 360 DNAArtificial Variable heavy chain region 1.0 21 gatgtacaac ttcaggagtcaggacctggc ctcgtgaaac cttctgagac cctgtctctc 60 acctgcgcag tcactggctactccatcacc agtggttatt actggagctg gatccggcag 120 tttccaggaa agaaactggaatggatgggc ttcataagct acgacggttc caataagtat 180 aatccatctc tcaaaaatcgaatctccatc actcgtgaca catctgagaa ccagtttttc 240 ctgaagttgt cttctgtgactgcagcagac acagcaacat attattgtgc aagaggttta 300 cgacgagggg actattctatggactactgg ggtcaaggaa ccctggtcac cgtctcctca 360 22 120 PRT ArtificialVariable Heavy chain region 1.0 22 Asp Val Gln Leu Gln Glu Ser Gly ProGly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala ValThr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Ser Trp Ile Arg GlnPhe Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Phe Ile Ser Tyr Asp GlySer Asn Lys Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr ArgAsp Thr Ser Glu Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val ThrAla Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Arg Arg GlyAsp Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr ValSer Ser 115 120 23 360 DNA Artificial Variable heavy chain region 2.0 23gatgtacaac ttcaggagtc aggacctggc ctcgtgaaac cttctgagac cctgtctctc 60acctgcgcag tcactggcta ctccatcacc agtggttatt actggagctg gatccggcag 120cctccaggaa agggcctgga atggatgggc ttcataagct acgacggttc caataagtat 180aatccatctc tcaaaaatcg aatctccatc actcgtgaca catctgagaa ccagtttttc 240ctgaagttgt cttctgtgac tgcagcagac acagcagtct attattgtgc aagaggttta 300cgacgagggg actattctat ggactactgg ggtcaaggaa ccctggtcac cgtctcctca 360 24120 PRT Artificial Variable heavy chain region 2.0 24 Asp Val Gln LeuGln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu SerLeu Thr Cys Ala Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr TrpSer Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly PheIle Ser Tyr Asp Gly Ser Asn Lys Tyr Asn Pro Ser Leu 50 55 60 Lys Asn ArgIle Ser Ile Thr Arg Asp Thr Ser Glu Asn Gln Phe Phe 65 70 75 80 Leu LysLeu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala ArgGly Leu Arg Arg Gly Asp Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 GlyThr Leu Val Thr Val Ser Ser 115 120 25 360 DNA Artificial Variable heavychain region 3.0 25 gatgtacaac ttcaggagtc aggacctggc ctcgtgaaaccttctgagac cctgtctctc 60 acctgcgcag tcagcggcta ctccatcacc agtggttattactggagctg gatccggcag 120 cctccaggaa agggcctgga atggatgggc ttcataagctacgacggttc caataagtat 180 aatccatctc tcaaaaatcg aatcaccatc tcccgtgacacatctaagaa ccagttttcc 240 ctgaagttgt cttctgtgac tgcagcagac acagcagtctattattgtgc aagaggttta 300 cgacgagggg actattctat ggactactgg ggtcaaggaaccctggtcac cgtctcctca 360 26 120 PRT Artificial Variable heavy chainregion 3.0 26 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys ProSer Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser IleThr Ser Gly 20 25 30 Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys GlyLeu Glu Trp 35 40 45 Met Gly Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr AsnPro Ser Leu 50 55 60 Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys AsnGln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr AlaVal Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Arg Arg Gly Asp Tyr Ser Met AspTyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 27360 DNA Artificial Variable heavy chain region 4.0 27 gatgtacaacttcaggagtc aggacctggc ctcgtgaaac cttctgagac cctgtctctc 60 acctgcgcagtcagcggcta ctccatcacc agtggttatt actggagctg gatccggcag 120 tttccaggaaagaaactgga atggatgggc ttcataagct acgacggttc caataagtat 180 aatccatctctcaaaaatcg aatcaccatc tcccgtgaca catctaagaa ccagttttcc 240 ctgaagttgtcttctgtgac tgcagcagac acagcaacat attattgtgc aagaggttta 300 cgacgaggggactattctat ggactactgg ggtcaaggaa ccctggtcac cgtctcctca 360 28 120 PRTArtificial Variable heavy chain region 4.0 28 Asp Val Gln Leu Gln GluSer Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu ThrCys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Ser TrpIle Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Phe Ile SerTyr Asp Gly Ser Asn Lys Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile ThrIle Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu SerSer Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly LeuArg Arg Gly Asp Tyr Ser Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr LeuVal Thr Val Ser Ser 115 120 29 324 DNA Artificial Variable light chainregion1.0 29 gacattcaga tgacccagtc tcaatctagt atgtccacat cagtaggagaccgagtcacc 60 atcacctgca aggccagtca gaatgtggtt acagctgtag cctggtatcgacagaaacca 120 ggaaagtctc ctaaactact gatttactcg gcatccaatc ggcacactggagtccctagt 180 cgcttctctg gcagtggatc tgggacagat ttcactctca ccatctctagcatgcagcct 240 gaagacttcg cagattattt ctgccagcaa tatagcagct atccgttcacgttcggaggg 300 gggaccaagc tcgagatcaa acgg 324 30 108 PRT ArtificialVariable light chain region 1.0 30 Asp Ile Gln Met Thr Gln Ser Gln SerSer Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys LysAla Ser Gln Asn Val Val Thr Ala 20 25 30 Val Ala Trp Tyr Arg Gln Lys ProGly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg His ThrGly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe ThrLeu Thr Ile Ser Ser Met Gln Pro 65 70 75 80 Glu Asp Phe Ala Asp Tyr PheCys Gln Gln Tyr Ser Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr LysLeu Glu Ile Lys Arg 100 105 31 324 DNA Artificial Variable light chainregion 2.0 31 gacattcaga tgacccagtc tccatctagt ctgtccgctt cagtaggagaccgagtcacc 60 atcacctgca aggccagtca gaatgtggtt acagctgtag cctggtatcgacagaaacca 120 ggaaagtctc ctaaactact gatttactcg gcatccaatc ggcacactggagtccctagt 180 cgcttctctg gcagtggatc tgggacagat ttcactctca ccatctctagcctgcagcct 240 gaagacttcg cagattattt ctgccagcaa tatagcagct atccgttcacgttcggaggg 300 gggaccaagg tcgagatcaa acgg 324 32 108 PRT ArtificialVariable light chain region 2.0 32 Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys LysAla Ser Gln Asn Val Val Thr Ala 20 25 30 Val Ala Trp Tyr Arg Gln Lys ProGly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg His ThrGly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe ThrLeu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Asp Tyr PheCys Gln Gln Tyr Ser Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr LysVal Glu Ile Lys Arg 100 105 33 324 DNA Artificial Variable light chainregion 3.0 33 gacattcaga tgacccagtc tccatctagt ctgtccgctt cagtaggagaccgagtcacc 60 atcacctgca aggccagtca gaatgtggtt acagctgtag cctggtatcagcagaaacca 120 ggaaaggccc ctaaactact gatttactcg gcatccaatc ggcacactggagtccctagt 180 cgcttctctg gcagtggatc tgggacagat ttcactctca ccatctctagcctgcagcct 240 gaagacttcg caacctatta ttgccagcaa tatagcagct atccgttcacgttcggaggg 300 gggaccaagg tcgagatcaa acgg 324 34 108 PRT ArtificialVariable light chain region 3.0 34 Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys LysAla Ser Gln Asn Val Val Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg His ThrGly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe ThrLeu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr TyrCys Gln Gln Tyr Ser Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr LysVal Glu Ile Lys Arg 100 105

What is claimed is:
 1. An antibody that competitively inhibits bindingof TMEFF2#19 to TMEFF2.
 2. The antibody of claim 1, wherein the antibodyis further conjugated to an effector component selected from the groupconsisting of a fluorescent label, a radioisotope and a cytotoxicchemical.
 3. The antibody of claim 2, wherein the cytotoxic chemical isauristatin.
 4. The antibody of claim 1, wherein the antibody is selectedfrom the group consisting of an antibody fragment, a humanized antibody,and TMEFF2#19.
 5. The antibody of claim 1, wherein the TMEFF2 is on acancer cell.
 6. A pharmaceutical composition comprising apharmaceutically acceptable excipient and the antibody of claim
 1. 7.The pharmaceutical composition of claim 6, wherein the antibody isfurther conjugated to an effector component selected from the groupconsisting of a fluorescent label, a radioisotope and a cytotoxicchemical.
 8. The pharmaceutical composition of claim 7, wherein thecytotoxic chemical is auristatin.
 9. The pharmaceutical composition ofclaim 6, wherein the antibody is a humanized antibody.
 10. Thepharmaceutical composition of claim 6, wherein the antibody isTMEFF2#19.
 11. A method of detecting a prostate cancer cell in abiological sample from a patient, the method comprising contacting thebiological sample with an antibody of claim
 1. 12. The method of claim11, wherein the antibody is further conjugated to a fluorescent label.13. A method of inhibiting proliferation of a prostate cancer-associatedcell, the method comprising the step of contacting the cell with anantibody of claim
 1. 14. The method of claim 13, wherein the antibody isan antibody fragment.
 15. The method of claim 13, wherein the prostatecancer cell is in a patient.
 16. The method of claim 14, wherein thepatient is a primate.
 17. The method of claim 16, wherein the patient isundergoing a therapeutic regimen to treat metastatic prostate cancer.18. The method of claim 16, wherein the patient is suspected of havingmetastatic prostate cancer.
 19. An antibody comprising SEQ ID NO:22, SEQID NO:24, SEQ ID NO:26, or SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, orSEQ ID NO:34.
 20. The antibody of claim 19, further conjugated to aneffector compound.
 21. The antibody of claim 19, wherein the antibodycomprises a protein encoded by SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID. NO:29, SEQ ID NO:31, and SEQ ID NO:33.
 22. Apharmaceutical composition comprising a pharmaceutically acceptableexcipient and the antibody of claim
 19. 23. A method of detecting acancer cell in a biological sample from a patient, the method comprisingcontacting the biological sample with an antibody of claim
 19. 24. Amethod of inhibiting proliferation of a prostate cancer-associated cell,the method comprising the step of contacting the cell with an antibodyof claim
 19. 25. A method of treating prostate cancer with an antibodyto TMEFF2, wherein said prostate cancer is selected from the groupconsisting of a primary prostate cancer, metastatic prostate cancer,locally advanced prostate cancer, androgen independent prostate cancer,prostate cancer that has been treated with neoadjuvant therapy, andprostate cancer that is refractory to treatment with neoadjuvanttherapy.